U.S. patent number 8,101,738 [Application Number 13/137,005] was granted by the patent office on 2012-01-24 for abfb-2 gene from penicillium funiculosum.
This patent grant is currently assigned to Adisseo France S.A.S.. Invention is credited to Jean Marie Francois, Olivier Nore, Jean-Luc Parrou, Olivier Tourrasse.
United States Patent |
8,101,738 |
Francois , et al. |
January 24, 2012 |
ABFB-2 gene from Penicillium funiculosum
Abstract
The invention relates to the abfB-2 gene of Penicillium
funiculosum that codes for a type B .alpha.-L-arabinofuranosidase
and has a cellulose binding domain. The enzyme
.alpha.-L-arabinofuranosidase can be incorporated in nutritional
additives or in foods for animals for which it improves the
digestibility and thus the nutritional value.
Inventors: |
Francois; Jean Marie
(Castenet-Tolosan, FR), Parrou; Jean-Luc (Toulouse,
FR), Tourrasse; Olivier (Toulouse, FR),
Nore; Olivier (Vernou sur Brenne, FR) |
Assignee: |
Adisseo France S.A.S. (Antony,
FR)
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Family
ID: |
35482299 |
Appl.
No.: |
13/137,005 |
Filed: |
July 14, 2011 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20110287139 A1 |
Nov 24, 2011 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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11918438 |
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8003114 |
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PCT/FR2006/000997 |
May 3, 2006 |
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Foreign Application Priority Data
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May 4, 2005 [FR] |
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05 04560 |
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Current U.S.
Class: |
536/23.2;
435/200; 426/62; 435/254.5; 536/23.7; 435/320.1; 536/23.4;
424/94.1 |
Current CPC
Class: |
C12Y
302/01055 (20130101); A23K 20/189 (20160501); C12N
9/2402 (20130101); Y10S 530/823 (20130101) |
Current International
Class: |
C07H
21/04 (20060101); A61K 38/43 (20060101); A23C
9/12 (20060101); C12N 1/00 (20060101); C12N
9/24 (20060101); C12N 15/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
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5863783 |
January 1999 |
Van Heuvel et al. |
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Foreign Patent Documents
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WO 99/57325 |
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Nov 1999 |
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WO |
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WO 2004/018662 |
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Mar 2004 |
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WO |
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Other References
Carvallo et al., "Characterization of an
.alpha.-L-arabinofuranosidase gene (abfl) from Penicillium
purpurogenum and its expression", Mycological Research, vol. 107,
No. 4 pp. 388-394, Apr. 2003. cited by other .
De Ioannes et al., "An .alpha.--arabinofuranosidase from
Penicillium purpurogenum:.production, purification and properties",
Journal of Biotechnology, vol. 76, No. 2-3, pp. 253-258, Jan. 21,
2000. cited by other .
Park et al., "A new method for the preparation of crystalline
-arabinose from arabinoxylan by enzymatic hydrolysis and selective
fermentation with yeast", Biotechnology Letters, vol. 23, No. 5,
pp. 411-416, Mar. 2001. cited by other .
Le Clinche et al., ".alpha.--Arabinofuranosidase from Aspergillus
terreus with Potential Application in Enology: Induction,
Purificaiton, and Characterization", Journal of Agricultural and
Food Chemistry, vol. 45, No. 7, pp. 2379-2383, Jul. 1997. cited by
other .
Hashimoto et al., ".alpha.--Arabinofuranosidase of Aspergillus
oryzae HL15", EMBL/GenBank/DDBJ databases, No. AB073860, Nov. 14,
2001. cited by other .
Hashimoto et al., ".alpha.--Arabinofuranosidase B",
EMBL/GenBank/DDBJ databases, No. Q96VA1, Dec. 1, 2001. cited by
other .
Margolles-Clark et al., "Cloning of Genes Encoding
.alpha.--Arabinofuranosidase and .beta.-Xylosidase from Trichoderma
reesei by Expression in Saccharomyces cerevisiae", Applied and
Environmental Microbiology, vol. 62, No. 10, pp. 3840-3846, Oct.
1996. cited by other .
Sakamoto et al., "Molecular characterization of a Penicillium
chrysogenum exo-1,5-.alpha.--arabinanase that is structurally
distinct from other arabinan-degrading enzymes", FEBS Letters, vol.
560, pp. 199-204 (2004). cited by other .
Koseki et al., "Role of Two .alpha.--Arabinofuranosidases in
Arabinoxylan Degradation and Characteristics of the Encoding Genes
from Shochu Koji Molds, Aspergillus kawachii and Aspergillus
awamori", Journal of Bioscience and Bioengineering, vol. 96, No. 3,
pp. 232-241 (2003). cited by other .
Gielkens et al., "The abfB gene encoding the major
.alpha.--arabinofuranosidase of Aspergillus nidulans: nucleotide
sequence, regulation and construction of a disrupted strain",
Microbiology, vol. 145, pp. 735-741 (1999). cited by other .
Brice et al., "The degradation of isolated hemiculluloses and
ligninhemicullulose complexes by cell-free, rumen hemicellulases",
Carbohydrate Research, vol. 101, pp. 93-100 (1982). cited by other
.
Panagiotou et al., "Induction, purification, and characterization
of two extracellular .alpha.--arabinofuranosidases from Fusarium
oxysporum", Canadian Journal of Microbiology, vol. 49, pp. 639-644
(2003). cited by other .
Flipphi et al., "Cloning and Characterization of the abfB Gene
Coding for the Major .alpha.--arabinofuranosidase (ABF B) of
Aspergillus niger," Curr. Genet, vol. 24, pp. 525-532, 1993. cited
by other.
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Primary Examiner: Devi; S.
Attorney, Agent or Firm: Berridge, PLC
Parent Case Text
This is a divisional of application Ser. No. 11/918,438 filed Apr.
21, 2008, now U.S. Pat. No. 8,003,114, which is the National Stage
Application of PCT/FR2006/000997 filed May 3, 2006, and claims the
benefit of French Application No. 05.04560 filed May 4, 2005. The
entire disclosures of the prior applications are hereby
incorporated by reference herein in their entirety.
Claims
What is claimed is:
1. An isolated polynucleotide encoding a polypeptide having
.alpha.-L-arabinofuranosidase B activity, wherein the
polynucleotide is selected from the group consisting of a
polynucleotide comprising a nucleotide sequence between position
268 and position 1470 of SEQ ID NO: 1 and a polynucleotide
comprising a nucleotide sequence between position 349 and position
1470 of SEQ ID NO: 1.
2. An isolated polynucleotide having the nucleotide sequence of SEQ
ID NO: 1 or the nucleotide sequence that is fully complementary to
the sequence of SEQ ID NO: 1.
3. An expression cassette comprising a promoter that is functional
in a host cell, a terminator sequence in the same host cell, and
the isolated polynucleotide according to claim 1.
4. A vector comprising the isolated polynucleotide of claim 1.
5. A vector comprising the expression cassette of claim 3.
6. An isolated host cell transformed with the isolated
polynucleotide of claim 1.
7. An isolated host cell transformed with the expression cassette
of claim 3.
8. An isolated host cell transformed with the vector of claim
4.
9. The isolated host cell of claim 6 wherein the host cell is
selected from the group consisting of yeasts and filamentous
fungi.
10. The isolated host cell of claim 9, wherein the host cell is a
Penicillium funiculosum strain.
11. A nutritional additive for animals comprising the host cell of
claim 6 and/or a fermentation broth of the host cell.
12. An expression cassette comprising a promoter that is functional
in a host cell, a terminator sequence in the same host cell, and
the isolated polynucleotide according to claim 2.
13. A vector comprising the isolated polynucleotide of claim 2.
14. A vector comprising the expression cassette of claim 12.
15. An isolated host cell transformed with the isolated
polynucleotide of claim 2.
16. An isolated host cell transformed with the expression cassette
of claim 12.
17. An isolated host cell transformed with the vector of claim
14.
18. The isolated host cell of claim 15, wherein the host cell is
selected from the group consisting of yeasts and filamentous
fungi.
19. The isolated host cell of claim 18, wherein the host cell is a
Penicillium funiculosum strain.
20. A nutritional additive for animals comprising the host cell of
claim 15 and/or a fermentation broth of the host cell.
Description
The invention relates to the abfB-2 gene isolated from Penicillium
funiculosum and the ABFB-2 polypeptide encoded by this gene having
an .alpha.-L-arabinofuranosidase B activity.
Penicillium funiculosum is a Talaromyces belonging to the
Aspergilleae family. The isolation of this microorganism from
numerous organic substrates which are subject to aerial or aqueous
contamination shows that this fungus possesses a range of
hydrolytic enzymes of a surprising richness. The use of this
enzymatic cocktail in animal feed contributes towards the
depolymerization of the natural organic substances and makes it
possible to improve their digestibility. WO 99/57325 thus describes
a Penicillium funiculosum strain called IMI378536 which produces a
mixture of enzymes which is particularly suitable as animal feed.
However, the enzymatic cocktails produced by Penicillium
funiculosum have not been biochemically characterized to any great
extent. Indeed, only a limited number of enzymatic activities, such
as xylanases and .beta.-glucanases are generally measured on the
fermentation broths obtained. These activities reflect only a
fraction of the enzymatic population present in the cocktail.
Hemicellulolytic compounds derived from agriculture constitute the
second polysaccharide reserve after cellulose in plant tissues.
This group is characterized by a wide variety of
heteropolysaccharides, of which the principle representatives are
xylans, arabinans, galactans, glucans and mannans. Arabinose, in
its furfural form, is widely represented among the
heteropolysaccharides such as arabinans and arabinoxylans. Arabinan
is a polymer with arabinofuranose residues linked by .alpha.-1-5
bonds and it may be substituted with 1 or 2 arabinose residues at
the O-2 or O-3 position. As regards the arabinoxylans, the
.alpha.-L-arabinofuranosyl residues are linked to the principle
.beta.-1-4-xylopyranosyl chain by .alpha.-1-3 and .alpha.-1-2
bonds. The presence of arabinose residues on these side chains can
restrict the enzymatic hydrolysis of hemicellulolytic compounds in
numerous industrial applications such as the enhancement of the
digestibility of animal feed. The enzymes cleaving the
.alpha.-L-arabinofuranoside bonds can act in synergy with xylanases
to allow the hydrolysis of arabinoxylans and arabinans.
The arabinase activities (endo-, exo-arabinase and, predominantly,
the .alpha.-L-arabinofuranosidase activities) can therefore
actively and synergistically contribute, with the xylanases, to the
depolymerization of the hemicellulolytic compounds. The
hemicellulolytic and pectic compounds may represent up to 50% of
the total carbohydrates present in plants and they constitute a
major source of energy for animals. The enhancement of the
digestibility of these compounds is correlated with the decrease in
the degree of substitution of the arabinosyl residues within the
hemicellulolytic compounds (Brice, R. E., Morrison, I. M. 1982,
Carbohydr. Res. 101: 93-100).
The enzymes which hydrolyse the bonds between L-arabinose residues
have been isolated from microorganisms such as bacteria or
filamentous fungi. Arabinosidases consist mainly of
.alpha.-L-arabinofuranosidases (EC 3.2.1.55) which are capable of
hydrolysing the non-reducing .alpha.-L-arabinofuranosyl residues
derived from L-arabinoxylan or compounds such as arabinans and
arabinogalactans.
The .alpha.-L-arabinofuranosidases (EC 3.2.1.55) have been
classified into two families of Glycoside Hydrolases (GH 51 and GH
54) according to their protein sequence similarities. These two
families differ by virtue of their specificity for substrate
contained in polysaccharides. The first group (GH 51) contains type
A arabinofuranosidases which act only on small linear structures of
.alpha.-1-5 linked arabinofuranosyl oligosaccharides. The second
group consists of type B arabinofuranosidases (GH 54) which
catalyse the hydrolysis of the .alpha.-1,5, .alpha.-1,3 and
.alpha.-1,2 bonds of the side chains contained in the
arabinofuranosyl-oligosaccharide compounds.
The B arabinofuranosidases (ABFB) have been isolated from numerous
bacteria, but also from filamentous fungi. The genus Aspergillus is
the most widely represented, but they have also been isolated from
the genera Trichoderma, Penicillium and Fusarium.
WO 96/29416, WO 96/06935 and U.S. Pat. No. 5,989,887 describe
Aspergillus niger arabinofuranosidase genes.
Gielkens et al. (Microbiology, 145, 735-741, 1999) have described
the Aspergillus nidulans abfB gene.
WO 96/29416, WO 96/06935, WO 2004/018662 and U.S. Pat. No.
5,989,887 describe Aspergillus niger arabinofuranosidase genes. The
protein sequence alignment indicates that the A. niger abfB protein
is 50.9% identical to the P. funiculosum ABFB-2 protein. None of
the characteristics essential for the use of the polypeptide in
animal nutrition is described in these applications.
Clinche et al. (J. Agric. Food Chem., 45, 2379-2383, 1997) have
described three .alpha.-L-arabinofuranosidases derived from
Aspergillus terreus having a potential application in oenology.
The Aspergillus kawachii and Aspergillus awamori abfB genes have
been described by Koseki et al. (J. of Bioscience and
Bioengineering, Vol. 96, No. 3, 232-241, 2003). These enzymes have
applications in the fermentation of the Japanese liquor shochu.
The abfB gene from the filamentous fungus Trichoderma reesei has
been described by Margolles-Clark et al. (Applied and Environmental
Microbiology, 3840-3846, 1996).
Panagiotou et al. have also described two extracellular
alpha-L-arabinofuranosidases derived from Fusarium oxysporum (Can
J. Microbiol. 2003: 49(10): 639-4).
Carvallo et al. (Mycol. Res., 107 (4), 388-394, 2003) have
described the B .alpha.-L-arabinofuranosidase from Penicillium
purpurogenum. The protein sequence alignment indicates that the P.
purpurogenum abf-1 protein is 51.2% identical to the P. funiculosum
ABFB-2 protein. None of the characteristics essential for the use
of the polypeptide in animal nutrition is described in this
article.
Sakamoto et al. (FEBS Letters 560, 199-204, 2004) have described
the Penicillium chrysogenum abnx gene encoding nevertheless an
arabinase activity distinct from the ABFB activity.
However, these ABFB enzymes do not have the optimum qualities
required for application in animal feed.
Indeed, to be utilizable in animal feed, the ABFBs must possess
properties compatible with the treatments to which the
feedingstuffs intended for this feed are subjected. In particular,
the activity of the enzymes used must be stable under the process
temperature and pH conditions, and, if possible, be optimum in the
preparation of these feedingstuffs and under the conditions which
exist in the digestive system of the animals ingesting these
feedingstuffs.
Furthermore, these enzymes must have a broad spectrum of action
(debranching) on the heteropolysaccharides (arabinans,
arabinoxylans and arabinogalactans) to allow effective enhancement
of the digestibility of the feedingstuffs by the animals. This
enhancement of the digestibility of the feedingstuffs makes it
possible to increase their nutritional value. Accordingly, the
enzymes having enhanced specificity (stereospecificity,
enantioselectivity), activity or affinity towards the natural
substrates arabinoxylans and arabinans are of great interest as
animal feed.
In addition, before hydrolysing the arabinofuranosyl bonds, it is
absolutely necessary beforehand to depolymerize Complex molecules
such as cellulose. The fungal cellulolytic enzymes (cellulases)
possess in general an fCDB domain (fungal type cellulose-binding
domain) which plays a predominant role in this depolymerization of
cellulose.
The present invention describes a Penicillium funiculosum
L-arabinofuranosidase B (ABFB-2) suitable for application in animal
nutrition and the gene encoding this enzyme. The invention also
relates to the homologues, variants and fragments of ABFB-2
preserving the same catalytic properties.
Advantageously, the ABFB enzymes according to the invention have a
very acidic optimum pH (pH 2.6) and preserve 55% of their activity
at pH 1.5.
Advantageously, the ABFB-2 enzyme possesses a fungal type
cellulose-binding domain (fCBD). This type of domain has been
described in other enzymes but had never been described for a
fungal arabinofuranosidase. It has been possible to experimentally
verify the functionality of this cellulose-binding domain of the
Penicillium funiculosum ABFB-2 enzyme. The presence of this binding
domain increases the affinity of the enzyme for its substrate and
consequently makes it possible to enhance the degradation of the
insoluble cellulose.
Thus, by virtue of its catalytic properties and its affinity for
cellulose, the Penicillium funiculosum ABFB-2 is therefore
particularly suitable for application as animal feed in
particular.
However, the enzymes according to the invention also have other
industrial or agroindustrial applications. There may be mentioned
in particular the treatment of fruit juices, the manufacture of
paper, the conversion of hemicellulolytic biomass to fuel or
chemical products, the preparation of alcoholic drinks by
fermentation.
Description of the Sequences
SEQ ID No. 1: Genomic sequence of the Penicillium funiculosum
abfB-2 gene.
SEQ ID No. 2: Sequence of the Penicillium funiculosum ABFB-2
polypeptide having type B .alpha.-L-arabinofuranosidase
activity.
SEQ ID No. 3: fCBD domain of the Penicillium funiculosum
ABFB-2.
SEQ ID No. 4: domain of low complexity of the Penicillium
funiculosum ABFB-2.
SEQ ID No. 5: fCBD domain of the Penicillium funiculosum cyanomyl
esterase.
SEQ ID No. 6: fCBD domain of the Penicillium funiculosum
endo-1,4-D-xylanase.
SEQ ID No. 7: fCBD domain of the Penicillium funiculosum xylanase
cellobiohydrolase.
SEQ ID No. 8: XbaI-abfB-2 PCR primer.
SEQ ID No. 9: HindIII-abfB-2 PCR primer.
DESCRIPTION OF THE INVENTION
The present invention relates to a polypeptide comprising a
polypeptide chosen from the following polypeptides: the polypeptide
of SEQ ID No. 2, the polypeptide whose sequence is between position
28 and position 400 of SEQ ID No. 2, a fragment of the polypeptide
of SEQ ID No. 2 having an .alpha.-L-arabinofuranosidase B
activity,
a polypeptide having an .alpha.-L-arabinofuranosidase B activity
and exhibiting at least 80% identity with the polypeptide of SEQ ID
No. 2.
The invention also relates to a polynucleotide, encoding an
.alpha.-L-arabinofuranosidase B activity, chosen from the following
polynucleotides: the polynucleotide whose sequence is comprised
between position 268 and position 1470 of SEQ ID No. 1, the
polynucleotide whose sequence is comprised between position 349 and
position 1470 of SEQ ID No. 1, a polynucleotide encoding a
polypeptide according to claim 1.
Another subject of the present invention is a polynucleotide having
the sequence represented by SEQ ID No. 1 or the sequence
complementary to SEQ ID No. 1.
The invention also relates to expression cassettes comprising, in
the direction of transcription: a promoter that is functional in a
host organism; a polynucleotide according to the invention; and a
terminator sequence that is functional in the same host
organism.
Another subject of the invention is a vector comprising a
polynucleotide according to the invention and/or an expression
cassette according to the invention.
The invention also relates to a host organism transformed with a
polynucleotide according to the invention, an expression cassette
according to the invention and/or a vector according to the
invention.
In one embodiment of the invention, the host organism is chosen
from yeasts and filamentous fungi.
Preferably, the host organism is a Penicillium funiculosum
strain.
The invention also relates to a nutritional additive for animals,
comprising a polypeptide according to the invention, a host
organism according to the invention or a fermentation broth of a
host organism according to the invention.
Preferably, this nutritional additive is in liquid form or in
powdered form.
Another aspect of the invention is a feedingstuff comprising a
nutritional base for animals and a nutritional additive for animals
according to the invention.
The invention also relates to the use of an ABFB polypeptide
according to the invention or a host organism according to the
invention for the manufacture of a nutritional additive for animals
or of a feedingstuff.
Another subject of the invention is the use of an ABFB polypeptide
according to the invention or of a host organism according to the
invention for hydrolysing the .alpha.-L-arabinofuranosyl bonds of
arabinofuranosyl-oligosaccharide compounds.
Polypeptides
The present invention therefore relates to ABFB polypeptides having
an .alpha.-L-arabinofuranosidase B activity. Preferably, these
polypeptides are isolated from Penicillium funiculosum.
The expression ".alpha.-L-arabinofuranosidase B" is understood to
mean .alpha.-L-arabinofuranosidases (EC 3.2.1.55) type B (GH 54)
which catalyse the hydrolysis of .alpha.-1,5, .alpha.-1,3 and
.alpha.-1,2 bonds of the side chains contained in
arabinofuranosyl-oligosaccharide compounds.
The polypeptides of the present invention are suitable for use in
animal nutrition.
The expression "polypeptide suitable for use in animal nutrition"
is understood to mean a polypeptide whose characteristics are such
that it is suitable for animal nutrition. The characteristics
essential for use in animal nutrition are in particular the pH and
the temperature at which the enzyme is active. Indeed, the pH of
the digestive system of the animals is acidic and it is therefore
essential that the enzyme remains active at this pH, this being in
order to preserve its activity in the hydrolysis of the L-arabinose
residues. In addition, conditioning the enzyme in a nutritional
additive or in the animal feed involves treatments and a
temperature greater than room temperature. The activity of the
enzymes used must therefore be stable under the process conditions,
and in particular the temperature conditions.
According to one embodiment of the present invention, the
polypeptide exhibits an .alpha.-L-arabinofuranosidase activity at
an acidic pH, for example less than 4.5, preferably less than 4.
Also, according to one embodiment of the present invention, the
polypeptide exhibits an optimum .alpha.-L-arabinofuranosidase
activity between pH 1.5 and pH 3.5.
According to a preferred embodiment of the present invention, the
polypeptide exhibits an .alpha.-L-arabinofuranosidase activity at
temperatures greater than room temperature. Preferably, the
polypeptide of the present invention has an optimum
.alpha.-L-arabinofuranosidase activity at a temperature of between
30.degree. C. and 70.degree. C., more preferably between 40.degree.
C. and 60.degree. C.
The .alpha.-L-arabinofuranosidase B of the Penicillium funiculosum
strain IMI378536 is represented in SEQ ID No. 2.
In a preferred embodiment, the polypeptides according to the
invention are glycosylated. The polypeptide of SEQ ID No. 2
possesses in particular N-glycosylation sites at amino acid 123 and
at amino acid 127. In a preferred embodiment, the asparagin
residues at position 123 and 127 of the polypeptide of SEQ ID No. 2
are glycosylated.
The .alpha.-L-arabinofuranosidase B of Penicillium funiculosum is
an enzyme secreted by the fungus into its extracellular
environment. The polypeptide of SEQ ID No. 2 thus comprises a
signal peptide of 27 amino acids. The subject of the invention is
also the mature polypeptide obtained after cleaving the signal
peptide. In particular, the invention relates to the polypeptide
whose sequence is between position 28 and position 400 of SEQ ID
No. 2. In another embodiment, the signal peptide of the polypeptide
of SEQ ID No. 2 may be replaced by a heterologous signal peptide
for the expression and the secretion of the polypeptide of SEQ ID
No. 2 by a heterologous host organism.
The invention also relates to fragments of the polypeptide of SEQ
ID No. 2 having an .alpha.-L-arabinofuranosidase B activity.
The term "fragment" of a polypeptide denotes a polypeptide
comprising a portion but not the entire polypeptide from which it
is derived. The invention thus relates to a polypeptide comprising
a fragment of at least 100, 200 or 300 amino acids of the
polypeptide of SEQ ID No. 2.
This fragment of the polypeptide of SEQ ID No. 2 preserves its
.alpha.-L-arabinofuranosidase activity. The invention therefore
relates to the biologically active fragments of the polypeptide of
SEQ ID No. 2. The term "biologically active fragment" denotes a
fragment of a polypeptide preserving the function of the
polypeptide from which it is derived. The biologically active
fragments of the polypeptide of SEQ ID No. 2 thus preserve the
function of the Penicillium funiculosum ABFB-2 polypeptide. These
biologically active fragments have an .alpha.-L-arabinofuranosidase
B activity. Preferably, the ABFB fragments according to the
invention possess a cellulose-binding domain. In a preferred
embodiment, the cellulose binding domain has the sequence described
in SEQ ID No. 3. Preferably, these fragments exhibit an optimal
.alpha.-L-arabinofuranosidase activity at pH 2.6.
The methods for preparing fragments of a polypeptide and the
techniques for measuring the .alpha.-L-arabinofuranosidase B
activity are well known to a person skilled in the art.
The subject of the invention is also polypeptides having an
L-arabinofuranosidase B activity and exhibiting at least 90%
identity with the polypeptide of SEQ ID No. 2. Preferably, these
polypeptides have the same properties and in particular the same
catalytic properties as the polypeptides of SEQ ID No. 2.
Preferably, these polypeptides are isolated from other strains of
Penicillium funiculosum or from other filamentous fungi.
Alternatively, these polypeptides may be obtained by site-directed
mutagenesis techniques for example.
The subject of the invention is polypeptides having at least 80%,
90%, 95%, 98% and preferably at least 99% of amino acids that are
identical with the polypeptide of SEQ ID No. 2.
The expression identical amino acids is understood to mean amino
acids that are invariant or unchanged between two sequences. These
polypeptides may exhibit a deletion, an addition or a substitution
of at least one amino acid compared with the polypeptide of SEQ ID
No. 2.
The subject of the invention is also polypeptides exhibiting at
least 90%, 95%, 98% and preferably at least 99% similarity with the
polypeptide of SEQ ID No. 2.
The expression similarity is understood to mean the measurement of
the resemblance between proteic or nucleic sequences. These
polypeptides may exhibit a deletion, an addition or a substitution
of at least one amino acid compared with the polypeptide of SEQ ID
No. 2. The degree of similarity between two sequences, quantified
by a score, is based on the percentage sequence identity and/or
sequence-preserving substitutions.
Methods for measuring and identifying the degree of identity and
the degree of similarity between polypeptides are known to persons
skilled in the art. It is possible to use for example Vector NTi
9.1.0, the alignment programme AlignX (Clustal W algorithm)
(Invitrogen INFORMAX). Preferably, the default parameters are
used.
The polypeptides according to the invention are isolated or
purified from their natural environment. The polypeptides may be
prepared by means of various methods. These methods are in
particular purification from natural sources such as cells
naturally expressing these polypeptides, the production of
recombinant polypeptides by appropriate host cells and their
subsequent purification, production by chemical synthesis or,
finally, a combination of these various approaches. These various
methods of production are well known to persons skilled in the art.
Thus, the ABFB polypeptides of the present invention may be
isolated from Penicillium funiculosum. In another embodiment, the
ABFB polypeptides of the present invention are isolated from
recombinant host organisms expressing an ABFB polypeptide according
to the invention.
The subject of the invention is also fusion proteins, recombinant
proteins or chimeric proteins comprising the polypeptides according
to the invention. The term "polypeptide" also denotes modified
proteins and polypeptides.
The polypeptides according to the present invention have an ABFB
activity. Preferably, the polypeptides according to the invention
possess a cellulose-binding domain. In a preferred embodiment, the
cellulose binding domain has the sequence described in SEQ ID No.
3. Preferably, the polypeptides exhibit an optimal
.alpha.-L-arabinofuranosidase B activity at pH 2.6. Preferably, the
polypeptides exhibit an optimal .alpha.-L-arabinofuranosidase B
activity at 50.degree. C.
Polynucleotides
The invention also relates to polynucleotides encoding an
.alpha.-L-arabinofuranosidase B activity. Preferably, these
polynucleotides encode a Penicillium funiculosum
.alpha.-L-arabinofuranosidase B.
According to the present invention, the expression "polynucleotide"
is understood to mean a single-stranded nucleotide chain or its
complementary DNA or RNA strand, or a double-stranded nucleotide
chain which may be of the complementary or genomic DNA type.
Preferably, the polynucleotides of the invention are of the DNA
type, in particular double-stranded DNA. The term "polynucleotide"
also denotes the modified polynucleotides.
The polynucleotides of the present invention are isolated or
purified from their natural environment. Preferably, the
polynucleotides of the present invention may be prepared by
conventional molecular biology techniques as described by Sambrook
et al. (Molecular Cloning: A Laboratory Manual, 1989) or by
chemical synthesis.
In a first embodiment, the invention relates to the polynucleotide
whose sequence is between position 268 and position 1470 of SEQ ID
No. 1. This polynucleotide encodes the Penicillium funiculosum
ABFB-2 enzyme of SEQ ID No. 2.
In a second embodiment, the invention relates to the polynucleotide
whose sequence is between position 349 and position 1470 of SEQ ID
No. 1. This polynucleotide encodes the Penicillium funiculosum
mature ABFB-2 polypeptide after cleavage of the signal peptide.
The invention also relates to polynucleotides having at least 70%,
75%, 80%, 85%, 90%, 95%, 98% and preferably at least 99% identity
with the polynucleotide whose sequence is between position 268 and
position 1470 of SEQ ID No. 1 and/or with the polynucleotide whose
sequence is between position 349 and position 1470 of SEQ ID No. 1.
These polynucleotides encode an .alpha.-L-arabinofuranosidase B
activity. Preferably, these polynucleotides encode a Penicillium
funiculosum .alpha.-L-arabinofuranosidase B.
The expression identical nucleotides is understood to mean
nucleotides that are invariant or unchanged between two sequences.
These polynucleotides may exhibit a deletion, an addition or a
substitution of at least one nucleotide compared with the reference
polynucleotide.
The invention also relates to polynucleotides exhibiting at least
70%, 75%, 80%, 85%, 90%, 95%, 98% and preferably at least 99%
similarity with the polynucleotide whose sequence is between
position 268 and position 1470 of SEQ ID No. 1 and/or with the
polynucleotide whose sequence is between position 349 and position
1470 of SEQ ID No. 1. These polynucleotides encode an
.alpha.-L-arabinofuranosidase B activity. Preferably, these
polynucleotides encode a Penicillium funiculosum
.alpha.-L-arabinofuranosidase B.
The expression similarity is understood to mean the measurement of
the resemblance between protein or nucleic sequences. These
polynucleotides may exhibit a deletion, an addition or a
substitution of at least one nucleotide compared with the reference
polynucleotide. The degree of similarity between two sequences,
quantified by a score, is based on the percentage sequence identity
and/or sequence-preserving substitution.
The methods for measuring and identifying the degree of identity
and the degree of similarity between nucleic acid sequences are
well known to persons skilled in the art. It is possible to use for
example Vector NTi Vector NTi 9.1.0, an alignment programme AlignX
(Clustal W algorithm) (Invitrogen INFORMAX). Preferably, the
default parameters are used.
Preferably, the polynucleotides exhibiting a degree of similarity
with a reference polynucleotide preserve the function of the
reference sequence. In the present case, the polynucleotides encode
an .alpha.-L-arabinofuranosidase B activity.
The invention also relates to polynucleotides capable of
selectively hybridizing with the polynucleotide whose sequence is
between position 268 and position 1470 of SEQ ID No. 1 and/or with
the polynucleotide whose sequence is between position 349 and
position 1470 of SEQ ID No. 1. Preferably, the selective
hybridization is carried out under conditions of average stringency
and preferably under conditions of high stringency. These
polynucleotides encode an .alpha.-L-arabinofuranosidase B activity.
Preferably, these polynucleotides encode a Penicillium funiculosum
.alpha.-L-arabinofuranosidase B.
The expression "sequence capable of selectively hybridizing" is
understood to mean, according to the invention, the sequences which
hybridize with the reference sequence at a level significantly
above the background noise. The level of the signal generated by
the interaction between the sequence capable of selectively
hybridizing and the reference sequences is generally 10 times,
preferably 100 times more intense than that of the interaction of
the other DNA sequences generating the background noise. Stringent
hybridization conditions allowing selective hybridization are well
known to persons skilled in the art. In general, the hybridization
and washing temperature is at least 5.degree. C. less than the Tm
of the reference sequence at a given pH and for a given ionic
strength. Typically, the hybridization temperature is at least
30.degree. C. for a polynucleotide of 15 to 50 nucleotides and at
least 60.degree. C. for a polynucleotide of more than 50
nucleotides. By way of example, the hybridization is carried out in
the following buffer: 6.times.SSC, 50 mM Tris-HCl (pH 7.5), 1 mM
EDTA, 0.02% PVP, 0.02% FICOLL, 0.02% BSA, 500 .mu.g/ml denatured
salmon sperm DNA. The washings are for example carried out
successively at low stringency in a 2.times.SSC, 0.1% SDS buffer,
at average stringency in a 0.5.times.SSC, 0.1% SDS buffer and at
high stringency in a 0.1.times.SSC, 0.1% SDS buffer. The
hybridization may of course be carried out according to other
customary methods well known to persons skilled in the art (see in
particular Sambrook et al., Molecular Cloning: A Laboratory Manual,
1989). Preferably, the polynucleotides selectively hybridizing with
a reference polynucleotide preserving the function of the reference
sequence. In the present case, the polynucleotides, which
selectively hybridize with the polynucleotide whose sequence is
between position 268 and position 1470 of SEQ ID No. 1 and/or with
the polynucleotide whose sequence is between position 349 and
position 1470 of SEQ ID No. 1, encode an
.alpha.-L-arabinofuranosidase B activity.
The invention generally relates to the polynucleotides encoding the
polypeptides according to the invention. Because of the degeneracy
of the genetic code, various polynucleotides can encode the same
polypeptide.
Another subject of the present invention is a polynucleotide whose
sequence is represented in SEQ ID No. 1. The polynucleotide of SEQ
ID No. 1 comprises sequences flanking the open reading frame (ORF)
of the Penicillium funiculosum abfB-2 gene. They are in particular
promoter and terminator sequences of the abfB-2 gene. The abfB gene
may be expressed from its homologous regulatory sequences, in
particular for overexpression in Penicillium funiculosum or in
other filamentous fungi.
In another embodiment, the abfB gene may be expressed in various
host organisms such as bacteria, yeasts and fungi for example. The
abfB gene may be expressed in a host organism under the control of
the promoter of SEQ ID No. 1 of the present invention or under the
control of a heterologous promoter.
Expression Cassettes
According to one embodiment of the invention, a polynucleotide
encoding a polypeptide according to the invention is inserted into
an expression cassette using cloning techniques well known to
persons skilled in the art. This expression cassette comprises the
elements necessary for the transcription and the translation of the
sequences encoding the polypeptides according to the invention.
Advantageously, this expression cassette comprises both elements
which make it possible to cause a host cell to produce a
polypeptide and elements necessary for the regulation of this
expression.
These expression cassettes comprise, in the direction of
transcription: a promoter that is functional in a host organism; a
polynucleotide according to the invention; a terminator sequence
that is functional in the same host organism.
Any type of promoter sequence may be used in the expression
cassettes according to the invention. The choice of the promoter
will depend in particular on the host organism chosen for the
expression of the gene of interest. Some promoters allow a
constitutive expression whereas other promoters are on the contrary
inducible. Among the promoters that are functional in fungi, there
may be mentioned in particular that for Aspergillus nidulans
glyceraldehyde-3-phosphate dehydrogenase (Roberts et al., Current
Genet. 15: 177-180, 1989). Among the promoters that are functional
in bacteria, there may be mentioned in particular that for the T7
bacteriophage RNA polymerase (Studier et al., Methods in enzymology
185: 60-89, 1990). Among the promoters that are functional in
yeasts, there may be mentioned the promoter for the GALL gene
(Elledge et al., Proc Natl Acad Sciences, USA. 88: 1731-1735, 1991)
or the S. cerevisiae GAL4 and ADH promoters. All these promoters
are described in the literature and are well known to persons
skilled in the art.
For expression in Penicillium funiculosum, expression cassettes
will be chosen for example that comprise a histone H4.B promoter,
an aspartyl acid protease promoter or a csl13 promoter (WO
00/68401).
The expression cassettes according to the present invention may
additionally include any other sequence necessary for the
expression of the polypeptides or polynucleotides, such as for
example regulatory elements or signal sequences allowing the
secretion of the polypeptides produced by the host organism. It is
possible to use in particular any regulatory sequence that makes it
possible to increase the level of expression of the coding sequence
inserted into the expression cassette. According to the invention,
it is possible to use in particular, in combination with the
regulatory promoter sequence, other regulatory sequences, which are
located between the promoter and the coding sequence, such as
transcription activators ("enhancer").
A wide variety of terminator sequences can be used in the
expression cassettes according to the invention, these sequences
allow the termination of transcription and the polyadenylation of
the mRNA. Any terminator sequence that is functional in the
selected host organism may be used.
For expression in Penicillium funiculosum, expression cassettes
will be chosen for example that comprise a histone H4.B terminator,
an aspartyl acid protease terminator or a csl13 terminator (WO
00/68401).
The subject of the present invention is also a polynucleotide
comprising an expression cassette according to the invention,
advantageously the expression cassettes according to the present
invention are inserted into a vector.
Vectors
The present invention therefore also relates to replicating or
expression vectors for transforming a host organism comprising at
least one polynucleotide or one expression cassette according to
the present invention. This vector may correspond in particular to
a plasmid, a cosmid, a bacteriophage or a virus into which a
polynucleotide or an expression cassette according to the invention
has been inserted. The techniques for constructing these vectors
and for inserting a polynucleotide of the invention into these
vectors are well known to persons skilled in the art. In general,
it is possible to use any vector capable of maintaining itself,
self-replicating or propagating in a host cell in order to induce
in particular the expression of a polynucleotide or of a
polypeptide. Persons skilled in the art will choose the appropriate
vectors according to the host organism to be transformed, and
according to the transformation technique used.
The vectors of the present invention are used in particular to
transform a host organism for replication of the vector and/or the
expression of a polypeptide according to the invention in the host
organism.
The invention also relates to a method for preparing a polypeptide
according to the invention comprising the following steps: a host
organism is transformed with an expression vector comprising an
expression cassette according to the invention and/or with a
polynucleotide according to the invention, the polypeptides
produced by the host organism are isolated. Host Organisms
The subject of the present invention is also a method for
transforming a host organism by integrating into the said host
organism at least one polynucleotide or an expression cassette or a
vector according to the invention. The polynucleotide may be
integrated into the genome of the host organism or can stably
replicate in the host organism. Methods for transforming the host
organisms are well known to persons skilled in the art and are well
described in the literature.
The present invention also relates to a host organism transformed
with a polynucleotide, an expression cassette or a vector according
to the invention. The expression host organism is understood to
mean in particular according to the invention any mono- or
pluricellular, lower or higher, organism, chosen from bacteria,
yeasts and fungi. The expression host organism is understood to
mean a non-human organism. Advantageously, the yeasts are chosen
from Pichia pastoris, Saccharomyces cerevisae, Yarrowia lipolytica
and Schwanniomyces occidentalis. The fungi are chosen from
Aspergillus and Penicillium, preferably from Penicillium
funiculosum, Trichoderma reesei, Aspergillus niger, Aspergillus
awamori, Aspergillus kawachii and Trichoderma koningii. In a
preferred embodiment, the host organism is a Penicillium
funiculosum strain in which an ABFB polypeptide according to the
invention is expressed or overexpressed.
The techniques for constructing vectors, transforming host
organisms and expressing heterologous proteins in these organisms
are widely described in the literature (Ausubel F. M. et al.,
"Current Protocols in Molecular Biology" Volumes 1 and 2, Greene
Publishing Associates and Wiley Interscience, 1989; T. Maniatis, E.
F. Fritsch, J. Sambrook, Molecular Cloning A laboratory Handbook,
1982).
Food Additives and Feedingstuffs
The present invention therefore relates to food additives providing
an .alpha.-L-arabinofuranosidase B activity. The intake of this
type of enzymatic activity makes it possible to enhance the
digestibility of the food and to increase its nutritional
value.
The expression nutritional additive is understood to mean a
substance that is intentionally added to a food, generally in small
quantities, in order to improve its digestibility or its
nutritional characteristics. The nutritional additives for animals
may contain for example vitamins, mineral salts, amino acids and
enzymes.
Typically, the nutritional additives for animals comprise a
polypeptide according to the invention, a host organism according
to the invention or a fermentation broth of a host organism
according to the invention. Thus, the polypeptides having an
.alpha.-L-arabinofuranosidase B activity according to the invention
can be purified or isolated from a Penicillium funiculosum strain
or from a recombinant host organism for the manufacture of a
nutritional additive for animals. Alternatively, a Penicillium
funiculosum strain or a host organism producing AbfB polypeptides
may be used directly for the manufacture of a nutritional additive
for animals. In a preferred embodiment of the invention, the
culture supernatant or fermentation broth of a Penicillium
funiculosum strain or of a host organism according to the invention
is used for the manufacture of nutritional additives for animals.
This embodiment is particularly advantageous when the ABFB
polypeptides are secreted by the Penicillium funiculosum strain or
the host organism. Usually, this culture supernatant is
concentrated or freeze-dried for the manufacture of the nutritional
additive.
Accordingly, the invention also relates to a method for preparing
an ABFB enzyme comprising the following steps: a) culturing a
Penicillium funiculosum strain or a transformed host organism
according to the invention under conditions for inducing the
expression of ABFBs, b) separating the culture supernatant
comprising the ABFB enzyme.
This culture supernatant or fermentation broth may then be
concentrated or freeze-dried for the formulation of a food additive
or of a feedingstuff.
If the host organism does not secrete the ABFB enzyme in the
culture medium, an additional step of opening the cells and
purifying the cellular extract may be necessary.
The nutritional additives of the present invention comprise an
.alpha.-L-arabinofuranosidase B activity but may also comprise
other nutritional substances such as vitamins, amino acids or
mineral salts.
The additives according to the invention increase the digestibility
of the feedingstuffs, thus contributing to a better enhancement of
the nutritional value of diets based on cereals (wheat, barley,
maize, oat, rye and the like) and on oilcakes (soybean, sunflower,
rapeseed and the like) in particular.
The present invention also relates to the feedingstuffs comprising
a nutritional base and a nutritional additive according to the
invention. These feedingstuffs are usually provided in the form of
meals or granules into which the additives according to the
invention are incorporated.
The expression feedingstuff is understood to mean anything that can
serve as food for animals.
The feedingstuffs comprise a polypeptide according to the
invention, a host organism according to the invention or a
fermentation broth of a host organism according to the
invention.
For intensive animal breeding, these feedingstuffs usually comprise
a nutritional base and nutritional additives.
The expression nutritional base is understood to mean what
constitutes the main part of the animal feed ration, consisting by
way of example of a mixture of cereals, proteins and fat of animal
and/or plant origin.
The nutritional bases for animals are suitable as feed for these
animals and are well known to persons skilled in the art. Usually,
these nutritional bases comprise, for example, maize, wheat, pea
and soybean. These nutritional bases are suitable for the needs of
the various animal species for which they are intended. These
nutritional bases may already contain nutritional additives such as
vitamins, mineral salts and amino acids.
In a preferred embodiment, the invention relates to feedingstuffs
for monogastric animals and in particular for poultry and pigs.
Poultry comprises in particular laying hens, broilers, turkeys and
ducks. Pigs comprise in particular growing-finishing pigs and
piglets.
DESCRIPTION OF THE FIGURES
FIG. 1: Organizational diagram of the fungal cellulose-binding
domain (fCBD).
FIG. 2: Determination of the optimum pH of the ABFB-2 enzyme in a
MCILVAINE buffer series (pH 2.2 to 8) at 40.degree. C. in the
presence of 5 mM PNPAF.
FIG. 3: Determination of the optimum temperature for the ABFB-2
enzyme at its optimum pH in the presence of 5 mM PNPAF.
FIG. 4: Determination of the kinetic constants K.sub.m and V.sub.m
(1/Vi=f(1/S) for ABFB-2 for a PNPAF range from 0.5 mM to 5 mM at pH
2.6 and 50.degree. C.
FIG. 5: Values of differential expression of the abfB-1 and abfB-2
genes according to the P. funiculosum growth conditions.
FIG. 6: Kinetics of the disappearance of ABFB-2 in the reaction
supernatant. The adhesion of the enzyme to the cell is monitored by
measuring the free residual .alpha.-L-arabinofuranosidase B
activity of ABFB-2.
EXAMPLES
Analysis of the Structure of the ABFB-2 Gene
ABFB-2 exhibits less than 62% identity with other ABFB enzymes of
filamentous fungi although these enzymes are in general fairly
conserved. This difference in identity is explained by the
identification of two distinct regions. In the N-terminal part, a
region specific for arabinofuranosidases (10 aa up to 341 aa) and
in the C-terminal region, a fungal type cellulose-binding domain
fCBD which was predicted by SMART (Simple Modular Architectural
Research Tool) analysis between position 367 and 400 aa. According
to the literature, this type of binding domain is generally found
in enzymes involved in the degradation of cellulose, such as
endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91)
(exoglucanases) or xylanases (EC 3.2.1.8). This is the first time
that a CBD domain has been found for an arabinofuranosidase.
From a structural (primary sequence) point of view, cellulases and
xylanases consist of two distinct domains, a catalytic domain and a
CBD domain linked by a so-called low complexity short sequence rich
in proline and/or in hydroxylated amino acid (Serine and
Threonine). The cellulose-binding domains have been studied in a
number of fungal cellulases, and they are characterized by a
sequence of up to 36 amino acids, present at the N-terminal end
(cbh-II or egl2) or C-terminal end of the protein (cbh-I, egl1 or
eg15). Furthermore, this type of domain is characterized by the
conservation of 4 cysteines according to FIG. 1 which allow the
formation of disulfide bridges.
The predicted fCBD domain for Penicillium funiculosum ABFB-2 is
composed of 34 amino acids including the 4 conserved cysteines (SEQ
ID NO:3).
This domain is preceded by a characteristic short sequence of 23
amino acids of low complexity (SEQ ID NO:4), rich in amino acids
bearing a hydroxyl (10 threonines and 6 serines). In Penicillium
funiculosum, this type of domain has been described in three other
enzymes, endo-1,4-xylanase, cellobiohydrolase and ferulic acid
esterase (cyanomyl esterase). The alignment of these four domains
confirms the conservation and the location of the 4 cysteines
within the fCBD.
TABLE-US-00001 496 529 AEFS_2 P. funiculosum (367)
THWGQCGGSGYSGPTSCVAPYACTTANPYYAQCL (SEQ ID NO: 3) cyanonamyl
esterase (320) AHWAQCGGIGYSGCTACASPYTCQKANDYYSQCL (SEQ ID NO: 5)
Endo 1-4 Xylanase D_P.f (374) AHWGQCGGIGWSGPTICVSPYTCQVLNPYYSQCL
(SEQ ID NO: 6) xylanase cellobiohydrolase (496)
AHWGQCGGQGWTGPTTCASGTTCCVVNPYYSQCL (SEQ ID NO: 7)
Alignment of the known fCBDs in Penicillium funiculosum
This type of binding domain has been widely described in
cellulolytic enzymes (cellulases). The analysis of the primary
sequence of fCBD by blast alignment, against known fCBD sequences,
identifies a very strong homology with the cellulose-binding
domains of cellobiohydrolase types I and II, and with the domains
of endoglucanases types I, II and V derived from Trichoderma reesei
and Aspergillus niger. The 3 tyrosine residues, and the asparagine
and glutamine residues, identified as being essential for the
functionality of the domain, are conserved within the ABFB-2
sequence. Studies of the structure/function relationship show that
this type of domain plays a predominant role in the
depolymerization of cellulose (Linder, M. & Teeri, T. T. (1997)
The roles and function of cellulose-binding domains, J. Biotechnol.
57 15-28) by increasing the binding affinity of the enzyme for its
substrate. In T. reesei, cellobiohydrolase I (CBH I) in the absence
of its fCBD domain exhibits a conserved catalytic activity, but the
binding affinity of the enzyme for cellulose is very substantially
reduced.
This is the first time that a binding domain of this type has been
identified in an arabinofuranosidase type B in a filamentous
fungus. The presence of this binding domain increases the affinity
of the enzyme for its substrate and consequently makes it possible
to enhance the degradation of insoluble cellulose.
Development of the Assay of the L-Arabinofuranosidase B
Activity
The L-arabinofuranosidase activity was measured from a P.
funiculosum culture on M2 medium with a mixed addition composed of
0.15% provasoy and 0.3% cellulose after 40 h. Samples were
collected at 48 h and 72 h of culture. The culture was performed in
a 200 ml Erlenmeyer flask with a useful volume of 50 ml. The
activity was determined by hydrolysing 5 mM
para-nitrophenyl-(L-arabinofuranoside (PNPAF) in a 50 mM sodium
acetate buffer, pH 5. 50 .mu.l of culture supernatant were
incubated with 250 .mu.l of substrate preheated at 50.degree. C.
for 15 min. The reaction was stopped by adding 500 .mu.l of 0.5 M
NaOH. The release of p-nitrophenyl (PNP) is measured at 405 nm with
a molar extinction coefficient of 17 000 M-1.cm.sup.-1. An enzyme
unit is defined as the quantity of enzyme which hydrolyses 1
.mu.mol of PNPAF per minute under the conditions described above.
For the culture of P. funiculosum, we obtained 20 mU.ml-1 after 48
h and 112 mU.ml-1 after 72 h of culture. These results are in
agreement with the literature, indeed for Aspergillus niger,
activities of the order of 100 to 600 mU.ml-1 were observed
according to the inducer used in the culture.
Cloning of P. funiculosum abfB-2 ORF and Transformation in
Saccharomyces cerevisiae
Starting with genomic DNA from P. funiculosum, the abfB-2 gene was
amplified by PCR with the aid of the pair of primers
(HindIII-abfB-2/XbaI-abfB-2). The following PCR conditions were
applied (94.degree. C. 30 sec; 62.degree. C. 30 sec; 1 min 30 sec
at 72.degree. C.) for 30 cycles. The PCR product of 1215 by was
cloned into a commercial vector pGEM-T(tm)easy.
Sequence of the PCR primer pair
XbaI-abfB-2: >5'-TCTAGAATGACGTCCAAACATAGTT-3'<(SEQ ID
NO:8)
HindIII-abfB-2: >5'-AAGCTTCTAGAGACATTGAGCGTA-3'<(SEQ ID
NO:9)
The HindIII/XbaI fragment of 1215 by was excised from the vector
pGEM-T and subcloned at the HindIII/XbaI sites into a shuttle
vector (plac195-PGK/CYC1). For heterologous expression, the abfB-2
gene is therefore under the control of the constitutive PGK
promoter of the gene encoding phosphoglycerate kinase (S.
cerevisiae) and the CYC1 terminator (S. cerevisiae) of the gene
encoding a cytochrome C oxidase activity. The new expression vector
is called pOT-02.
The S. cerevisiae strain JF #1194 (CEN.PK113-5D), a clone derived
from the strain CEN.PK 122 carrying the ura 3-52 auxotrophy, was
transformed (lithium acetate/heat shock method) with the expression
vector pOT-02. The transformant strains were selected by phenotype
complementation on uracil-free selective plates (URA3 marker).
Six transformants were selected in order to test for the presence
of an arabinofuranosidase B activity in the culture supernatant.
The transformants were cultured in 50 ml of uracil-free minimum
medium (except the wild-type control strain) for 24 hours. The
arabinofuranosidase activity was assayed on the culture
supernatants with the aid of the method described in the preceding
paragraph.
Determination of the Optimum pH
The abfB-2 gene encoding an arabinofuranosidase B activity derived
from P. funiculosum was cloned into S. cerevisiae. After checking
for the presence of an arabinofuranosidase B activity in several
transformants, a transformant was chosen and the ABFB activity was
assayed on the culture supernatant after 24 h of growth. The
cultures were carried out in a 200 ml Erlenmeyer flask (working
volume 50 ml). The activity was determined in the presence of 5 mM
p-nitrophenyl-.alpha.-L-arabinofuranoside (PNPAF) in a MCILVAINE
buffer series (pH 2.2 to 8.0). 80 .mu.l of culture supernatant were
incubated with 320 .mu.l of substrate preheated at 40.degree. C.
for 10 min. The reaction was stopped by adding 1 ml of 1M
Na.sub.2CO.sub.3. The release of p-nitrophenyl is measured at 405
nm.sup.-1. One enzyme unit is defined as the quantity of enzyme
which hydrolyses 1 gmol of PNPAF per minute. The activity curve is
represented in FIG. 2. ABFB-2 has an activity optimum at pH 2.6 and
preserves 55% of its activity at pH 3.8. This is the first time
that such a low optimum pH has been observed for an
arabinofurnasidase and that it has 68% activity at pH 1.5 in a 50
mH HCL solution.
Determination of the Optimum Temperature
Using the same protocol, we determined the optimum temperature for
the activity of ABFB-2. The enzyme was incubated for 10 min at each
of the temperatures in a MCILVAINE buffer at pH 2.6. The activity
curve is presented in FIG. 3.
An optimum temperature range for ABFBs is described in the
literature as being between 40 and 60.degree. C. The P. funiculosum
ABFB-2 has an activity optimum at 50.degree. C. If the optimum pH
and temperature of the enzyme are selected (pH 2.6 and 50.degree.
C.), it is observed that the activity for ABFB-2 is 20 times as
high as the activity determined in an acetate buffer pH 5 and
40.degree. C. (305 mU vs 15 mU).
Determination of K.sub.m and V.sub.m
For each of the two, the kinetic constants (K.sub.m and V.sub.m)
for ABFB-2 were determined by measuring the hydrolysis of PNPAF
over time, under the optimum conditions determined above.
The substrate (PNPAF) concentration ranges were established between
0.5 and 5 mM in a pH 2.6 buffer. The kinetics of hydrolysis was
monitored for 10 minutes at 50.degree. C. So as to obtain the
kinetic constants for both enzymes, the results were treated
according to the double inverse method (Lineweark and Burk) and are
presented in FIG. 4.
The kinetic constants were determined by hydrolysis of PNPAF under
optimum conditions. The Km value is 0.7 mM for ABFB-2. By
comparison, in the literature, the Km values for this type of
enzyme varies from 0.05 to 1.2 mM according to the genus and the
fungal species studied. ABFB-2 has a maximum speed of hydrolysis
(V.sub.m) of 328 mol PNPAF/mol of enzyme/min, under the conditions
described above.
Determination of the Molecular Weight of the ABFB-2 Enzyme
In order to determine the molecular weight of the ABFB-2 enzyme,
the culture supernatant, derived from the growth of a mutant (S.
cerevisiae) in a minimum medium, was concentrated 200-fold,
denatured by boiling at 100.degree. C. for 5 min, and then
deposited in an SDS-polyacrylamide gel.
It is observed that the quantity of extracellular proteins is
extremely low in the wild-type strain. For the mutants, the ABFB-2
enzyme is secreted into the culture supernatant. It is predominant
in relation to the basal level of the S. cerevisiae extracellular
proteins. The electrophonetic band obtained for the ABFB enzyme is
diffuse which suggests a strong glycosylation of the enzyme.
The determination of the molecular weight was carried out with the
aid of the size marker SEEBLUE (Invitrogen). The results are
presented in Table 1.
TABLE-US-00002 TABLE 1 ABFB-2 molecular weight in KDa Predicted MW
MW estimated on gel ABFB-2 41 55
We compared the molecular weight predicted by the algorithm Vector
NTi and the weight obtained by electrophoretic migration in a
denaturing SDS-PAGE gel. We observed an overestimation of the
molecular weight of the enzyme in the SDS-PAGE gel. A high
glycosylation of the enzyme is indeed suggested by the
visualization on gel of a diffuse electrophoretic band (O and N
glycosylations). The glycosylations occur during the processing of
the proteins in the expressing organism.
Analysis of the Profile of Expression of the abfB-2 Gene in
Penicillium funiculosum
Penicillium funiculosum possesses two genes encoding B
.alpha.-L-arabinofuranosidases: the abfB-1 and abfB-2 genes. The
profiles of expression of these genes under various P. funiculosum
culture conditions were compared.
P. funiculosum was cultured under conditions for inducing
cellulolytic and hemicellulolytid enzymes (type M2 industrial
growth medium) and under non-producing conditions (minimum glucose
medium M0). After 40 h of growth, the cultures were stopped, the
mycelium was recovered, and the total RNAs were extracted. The
quantity and the quality of the RNAs were assessed by measuring the
absorbance at 260 nm and at 280 nm (260/280 ratio>1.8). The
level of transcripts encoding the B-type arabinofuranosidase
(ABFB-1 and ABFB-2) activities were quantified under each of the
two conditions (M0 and M2) by real-time quantitative PCR.
The gene encoding P. funiculosum tubulin (tub-1) was used as a
control under the two conditions. This gene encodes a structural
protein that is essential for the integrity of the cell. This gene
is commonly used as the reference gene because it exhibits a
constant level of expression regardless of the culture condition
used (ubiquitous).
Specific primers for quantitative PCR were designed for each of the
genes (abfB-1, abfB-2 and tub-1). For both growth conditions (M0
and M2), 2 .mu.g of total RNA were retrotranscribed. A series of
dilutions of the complementary DNAs derived from the
retrotranscription were carried out in order to determine the
optimum conditions for amplification of the target genes
(constraints of the quantitative PCR method and for the efficiency
of these pairs of primers).
The normalized results are presented in Table 2 and FIG. 5.
TABLE-US-00003 TABLE 2 Values for differential expression of the
abfB-1 and abfB-2 genes as a function of the P. funiculosum growth
conditions M0 M2 abfb_1 1 107 abfb_2 1 1.27
The transcriptional regulations of the genes encoding cellulolytic
and hemicellulolytic activities have been described. The expression
of these genes is highly subject to the nature and/or to the
complexity of the carbon and nitrogen source on which the
microorganism is cultured. A high transcriptional repression of
these genes has been reported in the presence of glucose. This
regulation is performed via a catabolic repression protein CreA
which specifically binds to the promoter of these genes and blocks
their transcription. In our experiment for quantifying, by PCR, the
abfB-1 and abfB-2 messengers, that the level of expression of these
two genes under the glucose (M0) condition is very low. This is in
agreement with the literature since it has been shown that these
genes have a basal level of expression even under unfavourable
conditions (absence of cellulolytic and/or hemicellulolytic
substrates). The results obtained for the M0 condition are in
agreement with the literature. Unlike the abfB-1 gene, the
expression of the abfB-2 gene is not induced under industrial
conditions. This suggests that the enzymatic cocktails produced by
Penicillium funiculosum could be improved by obtaining the
expression of ABFB-2 by the fungus under industrial conditions or
by adding exogenous ABFB-2 to the enzymatic cocktail produced.
Determination of the Functionality of fCBD
To verify the functionality of the fCBD domain, tests for the
binding of the ABFB-2 enzyme were carried out on microcrystalline
cellulose.
The enzyme was mixed volume for volume with 0.5% of
microcrystalline cellulose in a 100 mM Tris-HCl buffer, pH 7.5 at
4.degree. C. The adhesion of the protein was monitored by measuring
the residual arabinofuranosidase B activity in the presence of
PNPAF under the optimal hydrolysis conditions determined. After
various periods of contact between the enzyme and the cellulose,
the mixture is centrifuged and the decrease in the concentration of
ABFB-2 protein is monitored by measuring the arabinofuranosidase B
activity in the supernatant. The binding tests were performed in
the presence of a control reaction containing the ABFB-2 enzyme,
under the same experimental conditions but in the absence of
microcrystalline cellulose. A second control was carried out by
incubating the ABFB-1 enzyme under the same reaction conditions in
the presence of microcrystalline cellulose. The results obtained
are presented in FIG. 6.
We can therefore observe that the fungal type cellulose-binding
domain (fCBD) of the enzyme ABFB-2 derived from P. funiculosum is
functional. The adhesion of the enzyme was monitored after 10 and
45 minutes of contact. After incubating for 10 minutes, 70% of the
total quantity of the enzyme initially present has adhered to the
microcrystalline cellulose. After 45 minutes, 80% of the entire
enzyme present in the reaction with cellulose has adhered. These
data suggest that the equilibrium of the contact reaction was
reached very rapidly and that the kinetics of adhesion of the
enzyme most certainly follows a kinetic law of order 1 relative to
the substrate within the first 10 minutes.
SEQUENCE LISTINGS
1
911639DNAPenicillium
Funiculosumpromoter(1)..(267)TATA_signal(167)..(172)CDS(268)..(1470)sig_p-
eptide(268)..(348)misc_binding(1366)..(1467)fCBD cellulose binding
domain 1ctagtgattg atcaagcacc atacccctct tccattctgg gataatatga
cggcatatac 60gttacatcag catacaatat agtcaaatta tcggttaaaa ggatgtattt
gaattatttg 120aattatccga ttttaaagac tccttcatag tgtgcgttag
aagcagtata aagcccgctt 180caatagctag cagttgaatc gatacccact
cgaacaacat ttttgaaacg caattgatac 240ttgtatagtc tcacaaaacg attcaac
atg acg tcc aaa cat agt ttc gaa cga 294 Met Thr Ser Lys His Ser Phe
Glu Arg 1 5gcc ggc ata ctt gca ttg ggc ctt att gct acg agc tct ctt
gtt gcc 342Ala Gly Ile Leu Ala Leu Gly Leu Ile Ala Thr Ser Ser Leu
Val Ala 10 15 20 25gcc ggc cct tgt gac atc tac tct tca ggt ggc aca
cca tgc gtt gcc 390Ala Gly Pro Cys Asp Ile Tyr Ser Ser Gly Gly Thr
Pro Cys Val Ala 30 35 40gcg cac agt acc act cgc gca ctc tat gat gct
tat act ggc ccg cta 438Ala His Ser Thr Thr Arg Ala Leu Tyr Asp Ala
Tyr Thr Gly Pro Leu 45 50 55tac caa gtg aca cgg agt tct gat agc agc
aag aaa gat atc gcg cca 486Tyr Gln Val Thr Arg Ser Ser Asp Ser Ser
Lys Lys Asp Ile Ala Pro 60 65 70ttg gcc gcc ggc ggc gtt gct aat gct
gcc acg caa gac tcc ttt tgt 534Leu Ala Ala Gly Gly Val Ala Asn Ala
Ala Thr Gln Asp Ser Phe Cys 75 80 85tca gga aca acc tgc ctc ata tct
atc atc tac gac caa tct gga aag 582Ser Gly Thr Thr Cys Leu Ile Ser
Ile Ile Tyr Asp Gln Ser Gly Lys 90 95 100 105ggg aac cat ctc acc
caa gct ccg aaa ggt ggc tgg agt gga cct gga 630Gly Asn His Leu Thr
Gln Ala Pro Lys Gly Gly Trp Ser Gly Pro Gly 110 115 120cca aat ggt
tca gat aat tta tcc agt gcg acc gcc gcc cca atc tat 678Pro Asn Gly
Ser Asp Asn Leu Ser Ser Ala Thr Ala Ala Pro Ile Tyr 125 130 135ctg
aac gga caa aag gcg tac ggc gtg ttt att gca cct ggt gac ggc 726Leu
Asn Gly Gln Lys Ala Tyr Gly Val Phe Ile Ala Pro Gly Asp Gly 140 145
150tac cgt aat gat aag act tct ggt ata gcc aca ggc gat caa ccc gag
774Tyr Arg Asn Asp Lys Thr Ser Gly Ile Ala Thr Gly Asp Gln Pro Glu
155 160 165gga atg tac gcc atc ttt gac gga acg cac tac aac ggc ggc
tgc tgc 822Gly Met Tyr Ala Ile Phe Asp Gly Thr His Tyr Asn Gly Gly
Cys Cys170 175 180 185ttc gac tac ggt aat gct gaa acc agt ggt acc
gat aca ggc gct ggc 870Phe Asp Tyr Gly Asn Ala Glu Thr Ser Gly Thr
Asp Thr Gly Ala Gly 190 195 200cac atg gag gct atc tac ttc ggt aac
tgt aat gtc tgg ggt tct ggt 918His Met Glu Ala Ile Tyr Phe Gly Asn
Cys Asn Val Trp Gly Ser Gly 205 210 215tct gga tca ggc cct tgg att
atg gct gat ttg gag aat ggt ctc ttc 966Ser Gly Ser Gly Pro Trp Ile
Met Ala Asp Leu Glu Asn Gly Leu Phe 220 225 230tcc ggt tat aac gcc
aaa caa aac acc gcc gat gca tcc atc aac tgg 1014Ser Gly Tyr Asn Ala
Lys Gln Asn Thr Ala Asp Ala Ser Ile Asn Trp 235 240 245cga ttc gtc
act gca att gtg aag ggc gag cca aac aat tgg gca atc 1062Arg Phe Val
Thr Ala Ile Val Lys Gly Glu Pro Asn Asn Trp Ala Ile250 255 260
265cgt ggt ggc aat gcc caa tct ggt tct ctc tcg aca tac tat aat ggc
1110Arg Gly Gly Asn Ala Gln Ser Gly Ser Leu Ser Thr Tyr Tyr Asn Gly
270 275 280ata cgc cca tca ggc tac aat ccg atg cac aaa gaa ggc gcc
att atc 1158Ile Arg Pro Ser Gly Tyr Asn Pro Met His Lys Glu Gly Ala
Ile Ile 285 290 295ctc ggc acg ggt ggt gac aac agt aac ggt gct caa
ggc act ttt tac 1206Leu Gly Thr Gly Gly Asp Asn Ser Asn Gly Ala Gln
Gly Thr Phe Tyr 300 305 310gag ggt gtg atg act tct ggg tac cct tct
gac tca act gag aat tcc 1254Glu Gly Val Met Thr Ser Gly Tyr Pro Ser
Asp Ser Thr Glu Asn Ser 315 320 325gtt caa gcc aat atc gtt gcc gcc
ggt tat tcc act tcg cct ggt agc 1302Val Gln Ala Asn Ile Val Ala Ala
Gly Tyr Ser Thr Ser Pro Gly Ser330 335 340 345cac acc act tcc acc
acc ctt acc acc atc act agt acc aca gca gta 1350His Thr Thr Ser Thr
Thr Leu Thr Thr Ile Thr Ser Thr Thr Ala Val 350 355 360tct gga gct
ggc cag aca cac tgg ggt cag tgt gga ggt agc gga tac 1398Ser Gly Ala
Gly Gln Thr His Trp Gly Gln Cys Gly Gly Ser Gly Tyr 365 370 375tcc
ggt cca acg agc tgt gtt gca ccc tac gct tgt aca acc gct aac 1446Ser
Gly Pro Thr Ser Cys Val Ala Pro Tyr Ala Cys Thr Thr Ala Asn 380 385
390cct tac tac gct caa tgt ctc tag aatataggcg ctcattcgtt ctatgactga
1500Pro Tyr Tyr Ala Gln Cys Leu 395 400agttggacaa gtatcaaaag
ctctctggag gcagggagtg tatttttgat gattataccg 1560tctggagaaa
gtgtatatag cttctcataa cccagacaat cagatatttc taacagagca
1620ataaatgaga gatgatcaa 16392400PRTPenicillium Funiculosum 2Met
Thr Ser Lys His Ser Phe Glu Arg Ala Gly Ile Leu Ala Leu Gly 1 5 10
15Leu Ile Ala Thr Ser Ser Leu Val Ala Ala Gly Pro Cys Asp Ile Tyr
20 25 30Ser Ser Gly Gly Thr Pro Cys Val Ala Ala His Ser Thr Thr Arg
Ala 35 40 45Leu Tyr Asp Ala Tyr Thr Gly Pro Leu Tyr Gln Val Thr Arg
Ser Ser 50 55 60Asp Ser Ser Lys Lys Asp Ile Ala Pro Leu Ala Ala Gly
Gly Val Ala 65 70 75 80Asn Ala Ala Thr Gln Asp Ser Phe Cys Ser Gly
Thr Thr Cys Leu Ile 85 90 95Ser Ile Ile Tyr Asp Gln Ser Gly Lys Gly
Asn His Leu Thr Gln Ala 100 105 110Pro Lys Gly Gly Trp Ser Gly Pro
Gly Pro Asn Gly Ser Asp Asn Leu 115 120 125Ser Ser Ala Thr Ala Ala
Pro Ile Tyr Leu Asn Gly Gln Lys Ala Tyr 130 135 140Gly Val Phe Ile
Ala Pro Gly Asp Gly Tyr Arg Asn Asp Lys Thr Ser145 150 155 160Gly
Ile Ala Thr Gly Asp Gln Pro Glu Gly Met Tyr Ala Ile Phe Asp 165 170
175Gly Thr His Tyr Asn Gly Gly Cys Cys Phe Asp Tyr Gly Asn Ala Glu
180 185 190Thr Ser Gly Thr Asp Thr Gly Ala Gly His Met Glu Ala Ile
Tyr Phe 195 200 205Gly Asn Cys Asn Val Trp Gly Ser Gly Ser Gly Ser
Gly Pro Trp Ile 210 215 220Met Ala Asp Leu Glu Asn Gly Leu Phe Ser
Gly Tyr Asn Ala Lys Gln225 230 235 240Asn Thr Ala Asp Ala Ser Ile
Asn Trp Arg Phe Val Thr Ala Ile Val 245 250 255Lys Gly Glu Pro Asn
Asn Trp Ala Ile Arg Gly Gly Asn Ala Gln Ser 260 265 270Gly Ser Leu
Ser Thr Tyr Tyr Asn Gly Ile Arg Pro Ser Gly Tyr Asn 275 280 285Pro
Met His Lys Glu Gly Ala Ile Ile Leu Gly Thr Gly Gly Asp Asn 290 295
300Ser Asn Gly Ala Gln Gly Thr Phe Tyr Glu Gly Val Met Thr Ser
Gly305 310 315 320Tyr Pro Ser Asp Ser Thr Glu Asn Ser Val Gln Ala
Asn Ile Val Ala 325 330 335Ala Gly Tyr Ser Thr Ser Pro Gly Ser His
Thr Thr Ser Thr Thr Leu 340 345 350Thr Thr Ile Thr Ser Thr Thr Ala
Val Ser Gly Ala Gly Gln Thr His 355 360 365Trp Gly Gln Cys Gly Gly
Ser Gly Tyr Ser Gly Pro Thr Ser Cys Val 370 375 380Ala Pro Tyr Ala
Cys Thr Thr Ala Asn Pro Tyr Tyr Ala Gln Cys Leu385 390 395
400334PRTPenicillium Funiculosum 3Thr His Trp Gly Gln Cys Gly Gly
Ser Gly Tyr Ser Gly Pro Thr Ser 1 5 10 15Cys Val Ala Pro Tyr Ala
Cys Thr Thr Ala Asn Pro Tyr Tyr Ala Gln 20 25 30Cys
Leu423PRTPenicillium Funiculosum 4Ser Thr Ser Pro Gly Ser His Thr
Thr Ser Thr Thr Leu Thr Thr Ile 1 5 10 15Thr Ser Thr Thr Ala Val
Ser 20534PRTPenicillium Funiculosum 5Ala His Trp Ala Gln Cys Gly
Gly Ile Gly Tyr Ser Gly Cys Thr Ala 1 5 10 15Cys Ala Ser Pro Tyr
Thr Cys Gln Lys Ala Asn Asp Tyr Tyr Ser Gln 20 25 30Cys
Leu634PRTPenicillium Funiculosum 6Ala His Trp Gly Gln Cys Gly Gly
Ile Gly Trp Ser Gly Pro Thr Ile 1 5 10 15Cys Val Ser Pro Tyr Thr
Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln 20 25 30Cys
Leu734PRTPenicillium Funiculosum 7Ala His Trp Gly Gln Cys Gly Gly
Gln Gly Trp Thr Gly Pro Thr Thr 1 5 10 15Cys Ala Ser Gly Thr Thr
Cys Thr Val Val Asn Pro Tyr Tyr Ser Gln 20 25 30Cys
Leu825DNAArtificial SequenceDescription of the Artificial Sequence
PCR Primer XbaI-abfB-2 8tctagaatga cgtccaaaca tagtt
25924DNAArtificial SequenceDescription of the Artificial Sequence
PCR Primer HindIII-abfB-2 9aagcttctag agacattgag cgta 24
* * * * *